Jung Jin Lim

Long-term proliferation and characterization of human spermatogonial stem cells obtained from obstructive and non-obstructive azoospermia under exogenous feeder-free culture conditions.

Objectives:  The aim of the present study was to improve efficiency of isolation and to optimize proliferative potential of human spermatogonial stem cells (SSCs) obtained from obstructive azoospermic (OA) and non‐obstructive azoospermic (NOA) patients, and further, to characterize these cells for potential use in infertility treatment or study of reproductive biology.

Identification of an intermediate state as spermatogonial stem cells reprogram to multipotent cells

The aim of this study was to understand the mechanisms that allow mSSC lines to be established from SSCs. Small, multilayer clumps of SSCs formed during two to four weeks of in vitro culture and were then transferred to MEF feeders. Small, round, monolayer colonies containing cells destined to convert to mSSCs, designated as intermediate state SSCs (iSSCs), first appeared after two to three passages. During an additional nine passages (47–54 days) under the same culture conditions, iSSCs slowly proliferated and maintained their morphology. Ultimately, a cell type with an ES-like morphology (mSSC) appeared from the iSSC colonies, and two mSSC cell lines were established. The mSSCs had a high proliferative potential in serum-free ES culture medium and have been successfully maintained since their first establishment (> 12 months). We also compared the specific characteristics of iSSCs with those of SSCs and mSSCs using immunocytochemistry, FACS, RT-PCR, DNA methylation, and miRNA analyses. The results suggest that iSSCs represent a morphologically distinct intermediate state with characteristic expression patterns of pluripotency-related genes and miRNAs that arise during the conversion of SSCs into mSSCs. Our results suggest that iSSCs could be a useful model for evaluating and understanding the initiation mechanisms of cell reprogramming.

In Vitro Culture-Induced Pluripotency of Human Spermatogonial Stem Cells

Unipotent spermatogonial stem cells (SSCs) can be transformed into ESC-like cells that exhibit pluripotency in vitro. However, except for mouse models, their characterization and their origins have remained controversies in other models including humans. This controversy has arisen primarily from the lack of the direct induction of ESC-like cells from well-characterized SSCs. Thus, the aim of the present study was to find and characterize pluripotent human SSCs in in vitro cultures of characterized SSCs. Human testicular tissues were dissociated and plated onto gelatin/laminin-coated dishes to isolate SSCs. In the presence of growth factors SSCs formed multicellular clumps after 2–4 weeks of culture. At passages 1 and 5, the clumps were dissociated and were then analyzed using markers of pluripotent cells. The number of SSEA-4-positive cells was extremely low but increased gradually up to ~ 10% in the SSC clumps during culture. Most of the SSEA-4-negative cells expressed markers for SSCs, and some cells coexpressed markers of both pluripotent and germ cells. The pluripotent cells formed embryoid bodies and teratomas that contained derivatives of the three germ layers in SCID mice. These results suggest that the pluripotent cells present within the clumps were derived directly from SSCs during in vitro culture.

Stem cell factor/c-Kit signaling in in vitro cultures supports early mouse embryonic development by accelerating proliferation via a mechanism involving Akt-downstream genes

PurposeStem cell factor (SCF)/c-Kit regulates the proliferation and survival of germ cells or stem cells; however, little is known about the role of SCF/c-Kit in pre-implantation embryo development.MethodsUsing exogenous SCF supplementation and c-Kit siRNA injection, we investigated the role and mechanism of SCF/c-Kit in pre-implantation mouse embryos.ResultsAddition of soluble SCF to the culture medium improved blastocyst formation. c-Kit gene silencing reduced the rate of blastocyst formation and delayed embryonic development. The number of proliferating cells in c-Kit gene-silenced blastocysts decreased, whereas the number of apoptotic cells in blastocysts obtained from both experimental and the control groups was not affected. RT-PCR, immunostaining and western blotting revealed that proliferation-related Akt downstream targets were substantially affected by c-Kit gene silencing.ConclusionSCF/c-Kit signaling through Akt downstream targets is likely involved in mediating the cleavage and proliferation of blastomeres during mouse pre-implantation embryogenesis.

Spermatogonial stem cell enrichment using simple grafting of testis and in vitro cultivation

Enrichment of spermatogonial stem cells (SSCs) from the mammalian adult testis faces several limitations owing to their relatively low numbers among many types of advanced germ cells and somatic cells. The aim of the present study was to improve the isolation efficiency of SSCs using a simple tissue grafting method to eliminate the existing advanced germ cells. Sliced testis parenchyma obtained from adult ICR or EGFP-expressing transgenic mice were grafted heterotropically under the dorsal skin of nude mice. The most advanced germ cells disappeared in the grafted tissues after 2–4 weeks. Grafted tissues were dissociated enzymatically and plated in culture dishes. During in vitro culture, significantly more SSCs were obtained from the grafted testes than from non-grafted controls, and the isolated SSCs had proliferative potential and were successfully maintained. Additionally, EGFP-expressing SSCs derived from graft parenchyma were transplanted into bulsufan-treated recipient mice testes. Finally, we obtained EGFP-expressing pups after in vitro fertilization using spermatozoa derived from transplanted SSCs. These results suggest that subcutaneous grafting of testis parenchyma and the subsequent culture methods provide a simple and efficient isolation method to enrich for SSCs in adult testis without specific cell sorting methods and may be useful tools for clinical applications.

Three-step method for proliferation and differentiation of human embryonic stem cell (hESC)-derived male germ cells.

The low efficiency of differentiation into male germ cell (GC)-like cells and haploid germ cells from human embryonic stem cells (hESCs) reflects the culture method employed in the two-dimensional (2D)-microenvironment. In this study, we applied a three-step media and calcium alginate-based 3D-culture system for enhancing the differentiation of hESCs into male germ stem cell (GSC)-like cells and haploid germ cells. In the first step, embryoid bodies (EBs) were derived from hESCs cultured in EB medium for 3 days and re-cultured for 4 additional days in EB medium with BMP4 and RA to specify GSC-like cells. In the second step, the resultant cells were cultured in GC-proliferation medium for 7 days. The GSC-like cells were then propagated after selection using GFR-α1 and were further cultured in GC-proliferation medium for 3 weeks. In the final step, a 3D-co-culture system using calcium alginate encapsulation and testicular somatic cells was applied to induce differentiation into haploid germ cells, and a culture containing approximately 3% male haploid germ cells was obtained after 2 weeks of culture. These results demonstrated that this culture system could be used to efficiently induce GSC-like cells in an EB population and to promote the differentiation of ESCs into haploid male germ cells.

Reversible infertility associated with testosterone therapy for symptomatic hypogonadism in infertile couple.

Purpose Androgen replacement therapy has been shown to be safe and effective for most patients with testosterone deficiency. Male partners of infertile couples often report significantly poorer sexual activity and complain androgen deficiency symptoms. We report herein an adverse effect on fertility caused by misusage of androgen replacement therapy in infertile men with hypogonadal symptoms. Materials and Methods The study population consisted of 8 male patients referred from a local clinic for azoospermia or severe oligozoospermia between January 2008 and July 2011. After detailed evaluation at our andrology clinic, all patients were diagnosed with iatrogenic hypogonadism associated with external androgen replacement. We evaluated changes in semen parameters and serum hormone level, and fertility status. Results All patients had received multiple testosterone undecanoate (NebidoR) injections at local clinic due to androgen deficiency symptoms combined with lower serum testosterone level. The median duration of androgen replacement therapy prior to the development of azoospermia was 8 months (range: 4-12 months). After withdrawal of androgen therapy, sperm concentration and serum follicle-stimulating hormone level returned to normal range at a median 8.5 months (range: 7-10 months). Conclusion Misusage of external androgen replacement therapy in infertile men with poor sexual function can cause temporary spermatogenic dysfunction, thus aggravating infertility.

Evaluation of sperm deoxyribonucleic acid (DNA) damage and effects on embryo development using a mouse cryptorchidism model.

OBJECTIVE To investigate the effects of sperm deoxyribonucleic acid (DNA) damage on fertilization and embryo development using a mouse cryptorchidism model of sperm DNA damage induction. MATERIALS AND METHODS Male ICR mice (aged 5-6 weeks) underwent cryptorchidism on their left testicles and sham operations on their right testicles. Spermatogenesis and sperm DNA fragmentation were assessed after 1, 2, and 4 weeks using hematoxylin-eosin staining, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling assays. Intracytoplasmic sperm injection into the oocytes of BDF1 females (aged 4-6 weeks) was performed using DNA-damaged sperm and normal sperm, and the fertilization rates and embryonic development were compared. RESULTS The testicular weight and size gradually decreased after induction of cryptorchidism, with progressive reduction of spermatogenesis and increased DNA damage after 1, 2, and 4 weeks. After intracytoplasmic sperm injection, the fertilization and blastocyst development rates were significantly lower in the cryptorchidism group; however, about one quarter of the embryos arising from DNA-damaged sperm continued to develop. CONCLUSION This was an in vivo animal study to evaluate the effects of sperm DNA damage using a cryptorchidism model. Sperm DNA damage increased significantly over time after cryptorchidism. This model could be useful in investigating male factor infertility and evaluating the biologic effects of paternal DNA damage on fertilization and future embryonic development.

DNA fragmentation of human sperm can be detected by ligation-mediated real-time polymerase chain reaction

OBJECTIVE To determine whether ligation-mediated real-time polymerase chain reaction (LM-RT-PCR), which combines LM-PCR, and RT-PCR, can detect sperm DNA fragmentation (DF) in human semen samples. DESIGN Three-way comparison of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), sperm chromatin dispersion (SCD), and LM-RT-PCR for detecting sperm DNA fragmentation. SETTING University hospital-based research laboratory. PATIENT(S) Twenty-five men presenting at an infertility clinic. INTERVENTION(S) Basic analysis of sperm concentration, motility, vitality, and morphology, with each semen sample equally divided into three aliquots that were evaluated for fragmentation using TUNEL, SCD, and LM-RT-PCR assays. MAIN OUTCOME MEASURE(S) In TUNEL and SCD assays, counts of the number of sperm with tetramethylrhodamine (TMR) red signals or no halo; in LM-RT-PCR results, evaluation of the threshold cycles (Ct) and relative fluorescence unit (RFU) values. RESULT(S) The median percentage of sperm with positive results for fragmentation in the TUNEL and SCD assays were 20.5% and 20.7%, respectively. To compare the accuracy of the TUNEL, SCD, and LM-RT-PCR assays, we divided the semen samples into two groups according to the TUNEL results: low and high percentage of sperm fragmentation. In the LM-RT-PCR results, the values of the cycles of threshold (Ct) and relative fluorescence unit (RFU) statistically significantly differed between the low and high percentage of sperm fragmentation groups. Comparisons among the TUNEL, SCD, and LM-RT-PCR assays revealed that the correlation patterns according to DNA fragmentation were similar in both the groups with high and low percentage of DNA fragmentation. Our morphologic analysis indicated that the fragmentation of sperm DNA did not appear to influence sperm morphology. CONCLUSION(S) These results indicate that the LM-RT-PCR technique is another useful tool for detecting DNA fragmentation, a parameter of sperm quality in human semen alone or combined with TUNEL or SCD assays.